Previous work from this laboratory has revealed the presence of an exon that can be spliced into the heavy chain of nonmuscle myosin II-B (NMHC II-B) in loop I near the ATP binding domain. Although NMHC II-B has a wide cellular distribution throughout the body, the inserted isoform is only expressed in neuronal cells. We now report that the newly recognized NMHC II-C also contains an alternative exon composed of 24 nucleotides encoding 8 amino acids. This exon, similar to that present in NMHC II-B, is spliced into loop I, but, unlike the neuronal-specific II-B isoform, is present in a wide variety of mouse tissues. These include liver, kidney and testis, where RT-PCR analysis reveals that the mRNA encoding the inserted form is greater than 90% compared to the noninserted isoform, as well as brain and lung, where it is about 50%. Adult mouse skeletal and cardiac muscle tissues are devoid of the inserted NMHC-IIC isoform, but human skeletal muscle NMHC II-C mRNA is more than 50% inserted isoform. The expression of the inserted isoform changes during mouse development, being 50% at embryonic stage 14 and rising to approximately 90% after birth in the liver and kidney. The mouse cerebrum contains less of the inserted isoform at E14 (approximately 20%), but increases to 50% one week after birth. On the other hand, mouse skeletal muscle myosin expresses over 90% of the inserted isoform at E14, which decreases to 0% after birth. A number of cell lines have been examined including mouse C2C12 (after differentiation, 100% noninserted isoform), rat PC12 (100% inserted) and Human HepG2 cells (100% inserted). The level of mRNA encoding the inserted isoform becomes higher than that of the noninserted isoform in RAW264.7 cell line treated with 2 mM Sodium Butyrate (SB) for one week. With 5mM SB, RAW 264.7 cells express only the inserted isoform and this expression level goes down with removal of SB from medium. We have cloned both isoforms of IIC fused with GFP protein to study their subcellular localization in a variety of cell lines.